In several neurologic disorders including Alzhemier disease, Parkinson disease, and diabetic neuropathy, axonopathy contributes significantly to morbidity and disease progression. Axon degeneration is an active self- destruct process by which compromised axons undergo rapid fragmentation initiated by a poorly-understood signaling cascade. To better understand this cascade, we developed a screening assay for compounds that delay fragmentation of transected mouse sensory axons in vitro. We used this screen to identify two kinases, IKK and GSK3, as probable regulators of axon degeneration. The proposed studies follow logically from this screen and are designed to demonstrate a link between each kinase and the mechanistic dismantling of axon cytoskeletal elements, a required step for axon self-fragmentation. The experiments outlined in this proposal will add to our limited understanding of how axons commit to self-destruction and may therefore inform therapeutic advances that reduce the burden of neurologic disease and injury. In Aim 1, we will test the hypothesis that IKK regulates Neurofilament breakdown in injured axons, as suggested by preliminary knockdown and pharmacologic studies. First, the dynamics of IKK activation will be studied in protein isolated from injured axons. We will assess whether IKK activation occurs subsequent to JNK and GSK3 activity using established inhibitors of each. Finally we will directly ask whether IKK is required for breakdown of Neurofilament protein in injured axons and whether Neurofilament removal involves IKK- dependent ubiquitination. In Aim 2, we will ask whether GSK3 contributes to axon degeneration by disrupting tau-microtubule interactions. First, we will use genetic ablation of GSK3 to determine whether it is required for normal axon degeneration as suggested by pharmacologic data. Next, because the critical phosphorylation site on tau, Thr231, mediates GSK3 disruption of tau-microtubule interactions, we will ask whether this site is becomes phosphorylated in injured axons and whether GSK3 inhibition blocks its phosphorylation. Finally, we will ask whether expression of non-phosphorylatable mutant tau - hypothesized to stabilize microtubules in the face of GSK3 activation - delays axon degeneration compared to wild-type tau. PUBLIC HEALTH RELEVANCE: Many nervous system diseases and injuries result in damage to axons - the delicate connections between nerve cells. For reasons not yet understood, damaged axons undergo a self-destruct process that may contribute to disease progression and worse clinical outcomes. This project will help us understand how damaged axons commit to self-destruction so that this process might be targeted by new therapies for nervous system diseases.